Clinical Significance of NKD Inhibitor of WNT Signaling Pathway 1 (NKD1) in Glioblastoma

Introduction As the most malignant type of gliomas, glioblastoma is characterized with disappointing prognosis. Here, we aimed to investigate expression and function of NKD inhibitor of Wnt signaling pathway 1 (NKD1), an antagonist of Wnt-beta-catenin signaling pathways, in glioblastoma. Methods The mRNA level of NKD1 was firstly retrieved from TCGA glioma dataset to evaluate its correlation with clinical characteristics and its value in prognosis prediction. Then, its protein expression level in glioblastoma was tested by immunohistochemistry staining in a retrospectively cohort collected from our medical center (n = 66). Univariate and multivariate survival analyses were conducted to assess its effect on glioma prognosis. Two glioblastoma cell lines, U87 and U251, were used to further investigate the tumor-related role of NKD1 through overexpression strategy in combination with cell proliferation assays. Immune cell enrichment in glioblastoma and its correlation with NKD1 level was finally assessed using bioinformatics analyses. Results NKD1 shows a lower expression level in glioblastoma compared to that in the normal brain or other glioma subtypes, which is independently correlated to a worse prognosis in both the TCGA cohort and our retrospective cohort. Overexpressing NKD1 in glioblastoma cell lines can significantly attenuate cell proliferation. In addition, expression of NKD1 in glioblastoma is negatively correlated to the T cell infiltration, indicating it may have crosstalk with the tumor immune microenvironment. Conclusions NKD1 inhibits glioblastoma progression and its downregulated expression indicates a poor prognosis.


Introduction
Glioma is the most common type of adult brain malignancies, which can be divided into astrocytoma, oligodendroglioma, oligoastrocytoma, and glioblastoma (GBM) according to the histological characteristics. Among them, GBM is the most malignant subtype and is also named as WHO grade IV glioma. Despite comprehensive treatment, the median overall survival time of GBM is only 12-16 months, which is far from satisfactory [1]. Terefore, detailed disease progression mechanisms and crucial biomarkers are essential for prognosis prediction and therapy development.
NKD inhibitor of Wnt signaling pathway 1 (NKD1) was frstly identifed in 2001 in both mouse and human, which has 10 exons and is located in chromosome 16q12 of humans [2,3]. NKD1 is reported to act as an antagonist of both the canonical and noncanonical Wnt-beta-catenin signaling pathways [4], thus participate in chicken embryonic development [5] although another study reported NDKs were dispensable for mice embryonic development [6].
As a negative feedback regulator of the Wnt signaling pathway, NKD1 is initially recognized with anticancer potentials. Mutation of NKD1 may result in a defcient at inhibiting Wnt signaling due to its disability to bind and destabilize Dishevelled (Dvl) proteins [7]. Abnormal expression of NKD1 has also been identifed in many malignancies. For example, low NKD1 enhances invasive capacity of NSCLC and correlates with unfavorable prognosis [8], while hypomethylation and high expression of NKD1 indicates better survival [9]. Downregulation of NKD1 was also correlated with a worse prognosis of breast invasive ductal carcinoma [10]. Consistent data were observed in acute myeloid leukemia, osteosarcoma, and uterine corpus endometrial carcinoma [11][12][13].
Nevertheless, the expression of NKD1 seems distinct in some other malignancies. For example, Arend Koch and his colleagues observed an elevated level of NKD1 in hepatoblastoma [14] although low NKD1 expression was observed in hepatocellular carcinoma [15]. Similarly, NKD1 overexpression was identifed in papillary predominant adenocarcinoma [16]. Furthermore, NKD1 was elevated in specifc mouse models of intestinal tumors comparing to healthy tissues, which represents a biomarker of tumor growth [17]. Moreover, NKD1 is highly expressed in colon carcinomas and enhances colon cancer growth according to both bioinformatics analyses and experimental validations [18].
Terefore, NKD1 shows distinct expression patterns in diferent tumor types. However, its expression and function in brain tumors remain unknown. Here, we tested the protein expression of NKD1 in GBMs for the frst time and revealed its clinical signifcance in predicting GBM prognosis. Moreover, we conducted bioinformatics analyses and cellular experiments to validate its tumor-related roles in GBM.

Patients and Samples.
We retrospectively enrolled 66 adult GBM patients who underwent surgical intervention in our hospital. All the specimens were confrmed as primary GBM according to pathological test. After exclusion, none of the patients had distant metastasis or previous malignant history at the time of diagnosis. In addition, patients who survived less than one month had been excluded. Te median age of enrolled patients was 35 years old, ranging from 18-80 years old. Te median follow-up time was 47.5 months, ranging from 1-93 months.

Immunohistochemistry (IHC)
Staining. IHC staining was performed to evaluate the protein expression level of NKD1 in GBM tissues. Formalin-fxedparafn-embedded specimens were cut into 4 μm slides, deparafnized and hydrated. Ten, slides underwent epitope retrieval using the heat-induced method in 90°C water for 1 hour. Ten, slides were treated with peroxidase to block endogenous reactions. Specifc anti-NKD1 primary antibody (1: 100 dilution) was used to incubate with mentioned slides overnight at 4°C. Te antibody used for IHC staining was rabbit polyclonal NKD1 antibody (ab185082, Abcam). On the next day, slides were subsequently incubated with HRP (horseradish peroxidase)linked secondary antibody and then underwent diaminobenzidine staining, followed by fnally counterstained with Hematoxylin. IHC images were independently assessed by two pathologists to distinguish the high-NKD1 expression or low-NKD1 expression of each specimen. Te fnal expression group was discussed by the two pathologists once there existed divergence.

Colony Formation.
Transfected cells were seeded into 6well plates at a density of 250 cells/well and cultured for 10 days at 37°C in a humidifed atmosphere containing 5% CO2. During the culturing, the medium was replaced every 4 days. After 10 days, formed colonies were fxed with methanol for 10 min, followed by crystal violet staining for another 15 min. Te colony numbers were counted and recorded.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Assay.
MTT strategy was introduced to evaluate the cell proliferation curve. Briefy, transfected cells were inoculated into a 96-well plate at 1000 cells/well in 100 μl volume. After culturing for 4 hours to allow cell adhesion, cells were further cultured for diferent designated time points. At each time point, 10 μl of MTT solution was added into each well and incubate for 3 hours in the incubator. Afterwards, the medium were removed and MTT crystals were solved. Finally, the absorbance was recorded at 550 nm using a microplate reader. Te experiment was conducted in triplicate and repeated three independent times.  [21]. P < 0.05 was defned as statistical signifcance. NS indicates no signifcance, * indicates P < 0.05, * * indicates P < 0.01, and * * * indicates P < 0.001.

Ethics. Tis study was approved by the Afliated Hospital of Inner Mongolia Medical University Ethic
Committee. Written informed consent was obtained from each participant.

NKD1-mRNA Level Is Higher in GBMs than Normal
Brains or Other Glioma Subtypes. We frstly analyzed the mRNA level of NKD1 in glioma tissues from the TCGA dataset. Accordingly, astrocytoma, oligoastrocytoma, and oligodendroglioma showed similar NKD1-mRNA level without statistically signifcant diference. However, GBM tissues contain signifcantly lower NKD1-mRNA level than the other three histological types previously (Figure 1(a), P < 0.001). Consistently, by dividing patients based on the WHO grade, we found that Grade IV GBM showed signifcantly a lower NKD1-mRNA level than Grade II or Grade III gliomas (Figure 1(b), P < 0.001). Interestingly, the NKD1-mRNA level was signifcantly lower in gliomas with wild type IDH than those with mutated IDH (Figure 1(c), P < 0.001). Tis was consistent with the previous fndings since most primary GBMs showed wild type IDH (Isocitrate dehydrogenase 1), while low-grade gliomas showed a higher mutated IDH rate [22]. It has been well-recognized that gliomas with 1p/19q codeletion possess better prognosis than those with noncodeletion [23], therefore, we next compared whether NKD1-mRNA show any expression diference in those two types. As a result, NKD1-mRNA level was signifcantly higher in 1p/19q codeletion specimens (Figure 1(d), P < 0.001), suggesting that high NKD1 may help predict a better prognosis. However, the TCGA dataset contains limited normal brain tissue samples; therefore, we also retrieved NKD1-mRNA information from the GTEx dataset to compare the diference between GBMs and normal brain tissues. As shown in Figure 1(e), NKD1-mRNA level was signifcantly downregulated in GBMs compared to that in normal brains (P < 0.001). Next, we analyzed whether NKD1 level has any prognostic signifcance for glioma prognosis (Figures 2(a)-2(c)). As expected, low NKD1 is signifcantly correlated with worse overall survival, cancer-specifc survival, and progress-free survival of glioma (all P < 0.001). In other words, lower-NKD1 mRNA level may predict poor glioma prognosis. Terefore, we were engaged to further investigate the prognostic role of NKD1-protein level in another retrospective cohort from our medical center.

Patients' Information.
Among the 66 enrolled surgicaltreated GBM patients in our medical center, there were 29 females and 37 males. Te entire diagnostic age was young with a median age of 35 years old, 36 cases were younger than 40 years old, while the other 30 cases were older. Among them, 6 patients showed tumor location in the parietal lobe, 24 cases in the temporal lobe, 32 cases in the frontal lobe, and the other 4 cases with unclear description about the detailed tumor location. Te median tumor size is 2.4 cm in diameter, ranging from 0.8-7.5 cm. According to personalized disease status, 37 cases underwent local resection, 12 cases underwent radical resection, while the other 17 cases underwent lobectomy. Till the end of follow-up, 18 cases were identifed as disease-specifc survival.

NKD1 Serves as a Novel Prognostic Factor for GBM.
We next conducted survival analyses based on each clinical variable ( Figure 3). As expected, elder patients exhibited worse prognosis than the younger ones (Figure 3(a), P � 0.006), while females and males showed no signifcant diference in cancer-specifc survival (Figure 3(b), P � 0.212). Although patients with parietal lobe tumor location seemed to had worse prognosis, the diference was not statistically signifcant (Figure 3(c), P � 0.053). Surprisingly, neither tumor size (Figure 3(d), P � 0.344) nor surgical pattern (Figure 3(e), P � 0.750) showed a signifcant efect on patients' survival perhaps due to limited case numbers.
As described in the method section, all the collected GBM tissue samples were subjected to IHC analyses to subgroup patients into low-NKD1 protein expression group and high-NKD1 protein expression group (Supplemental Figures S1A and S1B). Accordingly, 33 patients were characterized with low-NKD1 protein level, while the other 33 cases with high-NKD1 protein level. Survival analysis revealed that patients with low NKD1 protein levels in GBM samples exhibited signifcantly worse cancer-specifc survival than those with high-NKD1 protein levels ( Figure 3(f ), P � 0.027).
Te multivariate Cox regression model was further used to identify independent prognostic factors ( Table 1). As a result, elder age was identifed as an independent unfavorable factor (HR � 6.0, 95% CI 1.9-19.2, and P � 0.003), while frontal lobe tumor location was identifed as an independent favorable factor (HR � 0.2, 95% CI 0.1-0.7, and P � 0.011). Of note, a higher NKD1 expression level was also confrmed as an independent favorable prognostic factor of GBM for the frst time (HR � 0.3, 95% CI 0.1-0.8, and P � 0.019).

NKD1 Inhibits GBM Growth and Shows Cross-Talk with T Cell Infltration.
Since clinical evidence implied a potential tumor-suppressing role of NKD1, we next conducted cellular experiments to validate its detailed efects in GBM. NKD1 plasmids were transfected into U87 and U251 cells, respectively. RT-qPCR data confrmed the transfection effciencies compared to blank control and vector control (Figure 4(a)). Both the colony formation assay and MTT proliferation assay revealed an attenuated GBM growth after Genetics Research 3 overexpressing NKD1 (Figures 4(b) and 4(c)), highlighting the crucial role of NKD1 as a novel tumor suppressor. In addition, we analyzed the correlation between NKD1 level and immune cell enrichment (Figure 4(d)), which showed a negative correlation with T cells, neutrophils, and macrophages. For example, a lower mRNA level of NKD1 was signifcantly correlated with upregulated T cell infltration in GBM (Figure 4(e), P < 0.001), indicating the potential role of NKD1 in the immune environment during GBM progression.

Discussions
For the frst time, our data revealed a downregulated expression level of NKD1 in GBM on both mRNA and protein levels. RNA transcription and subsequent protein expression are negatively modulated by upstream methylation.
Consistently, high methylation of NKD1 CpG island is signifcantly correlated with a worse prognosis of epithelial ovarian cancer, which is independent from other clinical parameters according to multivariate Cox model analysis [24]. Similarly, a later study defned NKD1 methylation as an important unfavorable prognostic factor for a risk model of high-grade serous ovarian cancer [25]. NKD1 promoter was reported to be hypermethylated in U87 cell lines, however, its hypermethylation was not identifed in any gliomas (n � 70) according to Gotze's et al. data [26]. In contrast, NKD2-hypermethylation occurred in 43% (13/30) of the primary glioblastoma tissues, while a super low rate was observed in astrocytoma (1/30).
Besides methylation, we should also keep in mind that post-translational protein modifcations are critical for protein functions [27]. For example, the myristoylation of NKD2 is important for its plasma membrane localization  Genetics Research since its myristoylation-defcient mutant is only localized in cytoplasmic. Moreover, myristoylation of NKD2 antagonizes the Wnt-beta-catenin signaling pathway by degrading membrane-localized Dvl-1 [28]. Interestingly, Koch et al. reported that NKD1 in hepatoblastomas with betacatenin mutations had no antagonistic efect [14], which may result from specifc modifcations but require further investigations.
Clinical data indicated that low NKD1 level was signifcantly correlated with unfavorable GBM prognosis by both univariate and multivariate analyses. Moreover, we initially provided evidence that overexpressing NKD1 can signifcantly suppress the proliferation of GBM cells.
However, our study has several limitations. Firstly, we did not fully dig into the functional mechanisms of NKD1 in inhibiting GBM progression. Previous studies indicated that NKD1 interacts with Axin [29] and prevents nuclear accumulation of β-catenin [30], subsequently suppress Wnt signaling; whether NKD1 inhibits GBM growth through these mechanisms need detailed illuminations. Meanwhile, our data suggested that NKD1 expression was negatively correlated with T cell infltration; therefore, NKD1 may also be involved in the immunological microenvironment during GMB development. Secondly, our medical center has a limited GBM case number and we only enrolled 66 cases in our retrospective cohort to test NKD1 protein expression   level. Terefore, further evidence is necessary from more cases. Anyway, we believe our major conclusion because lower NKD1-mRNA level also indicates worse GBM prognosis in TCGA datasets, which is consistent with its protein signifcance in our cohort. Tirdly, recent studies suggested that NKD1 could serve as an independent predicting biomarker for tumor responsiveness of neoadjuvant chemo-radiotherapy in rectal cancer [31,32], and whether NKD1 can help direct chemotherapy of gliomas deserve further investigations.

Conclusions
NKD1 shows a decreased expression level in GBMs compared to normal brains or other glioma types, and its low expression results in poor GBM prognosis via enhancing GBM progression.

Data Availability
Data used to support the fndings of this study are available upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.